Concept

Dissolved molecules are transported with mobile phase through the HPLC column with stationery phase of opposing polarity. Sample components disperse and reach column end at different times – eluting as peaks.

Background

Application in:

• Purity & manufacturing control Analysis of drug – like molecules & toxins Separation & isolation of biopolymers Forensic & food industry

• Can use samples of liquid mixtures, ions, polymers, proteins with high MW.

• Sample must have right solubility in mobile phase, be particle free, stable and detectable.

• Classical liquid chromatography had column diameter in cm (not mm, as HPLC), low pressure, long time of analysis, soft packaging and glass columns – not steel; and used amount of sample in mg-g, not pg-μg.

• Reverse chromatography use polar mobile phase & non-polar stationery phase; minimalizing interactions between those.

  or hydrophobic SP & hydrophilic MP.

• Normal chromatography = polar SP & non-polar MP.

Mobile phase

• Used to transport the sample.

• Mix of water + organic solvent in low amount.

  • Organic solvent allow for the analyte to be eluted back to MP after SP interaction; reducing retention.

  • Polar MP organic solvents: methanol, ethanol;

  • Unpolar MP organic solvents: hexane, pentane.

  • Water is the weakest solvent due to high polarity & high retentions; while organic solvents are stronger due to lower polarity & reduced retentions.

• Isocratic – composition not changed during the analysis; 50% water, 50% ethanol.

• Gradient mode – composition changed over the run, increasing organic solvent amount for defined time and rate in reversed phase chromatography.

Stationery phase

• Usually RP C8 or RP C18; a silica gel with modified surface with carbon-hydrogen chains – the longer the chain, the less polar the phase.

• The number is the amount of C-H chains

• Less unpolar SP (C8) have lower affinity to the analytes; while very unpolar (C18) have high affinity towards analytes.

• The more bonds, the more hydrophobic it is; and the more the retention of hydrophobic analytes is decreased.

Setup

Solvent reservoir & binary pump provide constant flow ratio. Injection valve allow for defined sample introduction. Separation column & oven keep the right temperature, resolution and pressure. Detector of high sensitivity and large linear range; record dispersed band of solute as a peak – the greater the dispersion, the broader the peak – the more time the sample spends in the column – the more the bands spread – the more reduced the resolution is. Control units adjust parameters of separation & instrument. Sample plug starts as rectangle, then gets broader when in column.

Mechanism & calculations

• Columns have large surface to volume ratio; 80% is filled with solvent – 40% of pores, 40% of in-between; only 20% are the particles.

• Column volume V=πr^2 L r = column radius L = column length

• Analytes are transported via flow rate & separated by pore diffusion based on polarity.

• Mobile and stationery phases must differ in polarities, to show different interactions between molecules.

• Unpolar analytes have high affinity to unpolar stationery phase – would retain in the column as long, as MP is polar = longer.

• Analytes with greater affinity to MP will elute earlier.

• Distribution coefficient: molecules interact with MP & SP differently, having different distributions and net rates of migration.

• Column volume K=(Concentration in SP)/(Concentration in MP)

Chromatogram

• Retention time describes time from injection to detection (to peak max) – time in both phases.

• Dead time is the time in mobile phase.

  • Dead time markers have no interactions with SP as it doesn’t diffuse into pores;

  • thiourea for unpolar / hydrophobic SP;

  • n-hexane for polar / hydrophilic SP.

• Dead volume – in between particles

  Dead volume V_D=t_D×flow rate

• Peaks: Ideal when peak is straight

  • Fronting is shifting to the right; due to void column channels & too large sample volume.

    Column overloaded – inject less sample.

  • Tailing if shifting to the left; due to mass overload of sample, secondary analyte interactions on SP, column bed deformation.

• Resolution measure overlap between two peaks

  • RS < 1 strong overlap

  • RS = 1.5 identical area, 0.3% overlap

  • RS > 1.5 baseline separation

• Retention factor k standardized to dead time & independent of column.

  Compare basing on column dimensions and flow rate.

  • for unretained analyte k=0

  • for irreversible bound analyte k=∞

  • Recommended: 0.5 – 10 – above retention is strong, resolution & selectivity decreased. Selectivity α k=t_R-t_0

• Selectivity α described separation of peak max of 2 peaks.

  • High α = better relative separation of 2 peak maxima.

  • Recommended α > 1

  • To improve column selectivity changed should be MP, SP or MP pH.

• Number N of theoretical plates – imaginary column dividers – in those, analytes equilibrate between MP & SP.

  • Measure column efficiency, depends on substance and conditions.

  • The longer the N, the narrower the peaks, the better the efficiency.

Method development

The goal is to achieve the best resolution in shortest time by changing variables.

Start with MP composition (k & α), then SP and column conditions.

• Changing retention factor k between 0.5 – 20

  • increase resolution & analysis times;

• Changing selectivity α increase resolution by shifting peak maxima positions.

• Changing α change k.

• Factors influencing k & α:

  • MP & SP compositions

  • Temperature (small effect)

  • Solvent strength – increased would cause better separation due to changed chemical nature – retention time changed.

    Too much cause too short retention time so also overlap.

  • pH for ionizable analytes – change charge state of the peptides, changing the polarity

• Changing N – column conditions - increasing narrower peaks with less overlap; reducing dispersion Factors influencing N:

  • Flow rate

  • Column length

  • SP particle size

  • Column & packing materials

  • Column conditions improvement: by homogenous & spherical particles, small pores to get high surface area. Wide pores have reduced surface and sterical hindrance but separate macromolecules like proteins.

• Totally porous particles TPP

  • Most common & universal.

  • Give broad peaks due to different diffusion speeds through the particle.

• Superficially porous particles SPP

  • Solid core with thin porous shell; give shorter diffusion paths, cause sharp peaks for higher sensitivity, limit sample loads.

• Perfusion particles

  • Small diffusion peak with large pores. No diffusion, flow by flow rate.

Packaging based on silica materials SiO2:

• High mechanical strength – sustainable

• High column efficiency due to tailored TPP with different pore sizes

• Chemical surface able to be modified with functional groups

• Compatible with aqueous & organic solvents

• No swelling

• Stable in pH 3 – 9, degraded by hydrolysis

• Polar, but chlorosilanes make unpolar for reverse phase.

• Endcapping of free silanol groups with short alkyl residues to reduce surface silanol population (blocking silanol group – analyte interactions)

Hybrid silica material:

• methyl group replace surface silanol (give stability),

• bond susceptible to pH hydrolysis,

• endcapped silanol group.

• Stable in high pH

• Lower activity with basic analytes; less tailing Improved peak shape

• Limited surface area due to limited porosity

Packaging based on polymer-based materials:

• Stable in pH 1 – 13

• Have silanol groups, acidic; that make basic analytes make peaks with tailing.

• LC-MS required water wettable phases to ionise the analytes and make them less hydrophobic.

  • If it is more than 95% water, the phase collapse.

  • Are small, organic acid molecules with hydrophobic & basic properties.

  • Control spacing between bonded ligands – wide them

  • Add selectivity of low energy silanol groups

  • Vicinal silanol groups are hydrated

• Ligands are used for hydrophobic & hydrophilic interactions with analyte, prevent phase collapse.

  • Have mixed mode of retention and are compatible with LC-MS.

• Polar embedded phases

  • prevent collapse of C18 ligands

  • provide selectivity (due to hydrophilic interactions)

  • can be used in 100% aqueous MP.

Ionic analytes in rp-HPLC – ipc

A way to handle strongly ionic compounds in RP-HPLC.

• Retention times of basic & acidic analytes in RP-HPLC depend on the pH value of MP; but the pKa of the analyte is also important.

• If pH is equal to pKa, 50% of the molecules will be charged and 50% uncharged = lead to broad peak shapes due to equilibrium & make difficult reproducibility;

  Low pH = most retention;

  High pH = least retention; 50:50 occurs in unbuffered aqueous MP – require buffers with pKa equal to highest capacity pH.

Method development:

1. pH adjusted so the analyte is uncharged; (Acidic require acidic pH; pH < analyte pKa; Basic require alkaline pH; pH > analyte pKa).

2. Difference between pH of MP & pKa of analyte is at least one unit; making 90% uncharged analyte

3. Choose suitable buffer for pH: max buffer capacity if pH = buffer pKa

   Higher buffer capacity required total buffer concentration between 10-100mM.

Concept

• Ion pair chromatography IPC use RP columns to separate charged analytes dissolved in MP.

• Allows to simultaneously separate ionised & non-ionised analytes.

• Ion pair reagents have hydrophobic & hydrophilic parts (amphiphilic structure)– are surfactants.

• Ion pair use opposite charge to that of ionic analyte to produce uncharged ion pair at the end.

• Partition model: analyte molecule interacts first with ion pair reagent in the MP.

• Adsorption model: ion pair reagent react with surface of SP first. First the pH is adjusted so the analyte is fully ionised: acidic analytes require pH > pKa, basic analytes require pH < pKa.

  Retention time increase with increased concentration of ion pair reagent & with increased alkyl chain length of ion pair reagent.

• Problem: peak tailing due to uncontrolled ionic interactions with ionised silanol groups at pH > 6; between + charged analytes & - charged analytes. Reduced with Endcapping, pH change (remove ionisation), increased buffer concentration (mask silanol interactions), modify MP with TEA (interact with ionised silanol instead of analyte). Tailing factor T_f=W(0.05)/2f

Reverse phase chromatography

• Non-polar SP, polar MP

• Separation principle is the retention based on hydrophobicity, used for bioorganic molecules (peptides, proteins).

• Eluotropic series have the solvent strength adjusted with water & co-solvents (methanol, ACN) to change retention & selectivity. Used to optimise NPC.

  • Lower viscosity = higher separation, lower pressure.

  • UV absorption < 190 nm Toxic THF as co-solvent

  • Minor polarity, miscible with water

  • Slow column regeneration

  • Reactive with oxygen

  • Critical resolution on chromatogram describes resolution between two worst resolved peaks.

• Typical MP is water + methanol / CAN

• Selectivity for non-ionic samples depends on:

  • Mobile phase composition (best way)

• Stationary phase is unpolar bonded phases on silica support or polymer based.

Gradient hplc

Mobile phase is modified during the run by increasing solvent strength over time (reducing water content). Allow for shorter analysis times while retaining baseline separations.

Used for samples with wide k-value ranges & for protein samples (improved reproducibility for proteins); at higher % proteins are denaturated = improved reproducibility for proteins. Softening the gradient = higher resolution + high retention time.

• Method development:

  • start with fast-linear gradient with max. 6 column types. Best combination determined by most peaks & their shape.

  • Then modifications of organic solvent in MP as a gradient;

  • lastly eluents combination,

  • pH,

  • column types,

  • temperature,

  • gradient steepness,

  • temperature,

  • column length.

Normal phase chromatography npc

Opposite to RP-HPLC: works with unpolar MP (hexane, isooctane) and polar SP (silica beads, polar bonded phases on silica support). Produce opposite to RP-HPLC elution orders due to opposite polarity. Used for bioanalytical application – separation of fat-soluble vitamins. Sample should be insoluble in aqueous systems, unretained / too strongly retained by RP-HPLC, do unsatisfactory band spacing with RP-HPLC Optimisation with columns of smaller particle diameters (for higher retention factors), solvent strength based on eluotropic series for NPC: (tert. butanol > toluene > isooctane > n-hexane); solvent strength cannot be too high (polar solvents interact strongly with surface of SP) Band broadening Sample plug starts with rectangular shape and broadener & dilute during separation. When getting broader theoretical plate increase. Van- Deemter equation H=A+B/u+C×u H = height, measure band broadening, equal to N u = linear flow rate [cm/s] L = column length N = theoretical plates H=L/N Contributing factors: A-term: flow rate independent, describes Multipath effect (Eddy diffusion). Describes how molecules of the same kind have different pathways through the column due to particle size & shape; giving different retention times. Use homogenous column beds with smaller & spherical beads to counteract. B-term: pronounced at lower flow rates, described longitudinal diffusion. Plug diffuses parallel to the column axis becoming broaden. Counteracted by higher flow rate & solvent viscosity. C-term: pronounced at higher flow rates, described mass transfer between MP & SP. Strong at higher flow rates as the analytes have no time to interact with SP particles = less time to average the differences. Counteracted by small TPP or core-shell particles for shorter diffusion times; higher temperature to give low viscosity and increase mass transfer; lowering flow rate.

Dissolved molecules are transported with mobile phase through the HPLC column with stationery phase of opposing polarity. Sample components disperse and reach column end at different times – eluting as peaks.

Background Application in: Purity & manufacturing control Analysis of drug – like molecules & toxins Separation & isolation of biopolymers Forensic & food industry

Can use samples of liquid mixtures, ions, polymers, proteins with high MW.

Sample must have right solubility in mobile phase, be particle free, stable and detectable. Classical liquid chromatography had column diameter in cm (not mm, as HPLC), low pressure, long time of analysis, soft packaging and glass columns – not steel; and used amount of sample in mg-g, not pg-μg. Reverse chromatography use polar mobile phase & non-polar stationery phase; minimalizing interactions between those. or hydrophobic SP & hydrophilic MP. Normal chromatography = polar SP & non-polar MP. Mobile phase Used to transport the sample. Mix of water + organic solvent in low amount. Organic solvent allow for the analyte to be eluted back to MP after SP interaction; reducing retention. Polar MP organic solvents: methanol, ethanol; Unpolar MP organic solvents: hexane, pentane. Water is the weakest solvent due to high polarity & high retentions; while organic solvents are stronger due to lower polarity & reduced retentions.

Isocratic – composition not changed during the analysis; 50% water, 50% ethanol. Gradient mode – composition changed over the run, increasing organic solvent amount for defined time and rate in reversed phase chromatography. Stationery phase Usually RP C8 or RP C18; a silica gel with modified surface with carbon-hydrogen chains – the longer the chain, the less polar the phase. The number is the amount of C-H chains Less unpolar SP (C8) have lower affinity to the analytes; while very unpolar (C18) have high affinity towards analytes. The more bonds, the more hydrophobic it is; and the more the retention of hydrophobic analytes is decreased.

Setup

Solvent reservoir & binary pump provide constant flow ratio. Injection valve allow for defined sample introduction. Separation column & oven keep the right temperature, resolution and pressure. Detector of high sensitivity and large linear range; record dispersed band of solute as a peak – the greater the dispersion, the broader the peak – the more time the sample spends in the column – the more the bands spread – the more reduced the resolution is. Control units adjust parameters of separation & instrument. Sample plug starts as rectangle, then gets broader when in column.

Mechanism & calculations Columns have large surface to volume ratio; 80% is filled with solvent – 40% of pores, 40% of in-between; only 20% are the particles. Column volume V=πr^2 L r = column radius L = column length Analytes are transported via flow rate & separated by pore diffusion based on polarity. Mobile and stationery phases must differ in polarities, to show different interactions between molecules. Unpolar analytes have high affinity to unpolar stationery phase – would retain in the column as long, as MP is polar = longer. Analytes with greater affinity to MP will elute earlier. Distribution coefficient: molecules interact with MP & SP differently, having different distributions and net rates of migration. Column volume K=(Concentration in SP)/(Concentration in MP)

Chromatogram Retention time describes time from injection to detection (to peak max) – time in both phases. Dead time is the time in mobile phase. Dead time markers have no interactions with SP as it doesn’t diffuse into pores; thiourea for unpolar / hydrophobic SP; n-hexane for polar / hydrophilic SP. Dead volume – in between particles Dead volume V_D=t_D×flow rate Peaks: Ideal when peak is straight Fronting if shifting to the right; due to void column channels & too large sample volume. Column overloaded – inject less sample. Tailing if shifting to the left; due to mass overload of sample, secondary analyte interactions on SP, column bed deformation. Resolution measure overlap between two peaks RS < 1 strong overlap RS = 1.5 identical area, 0.3% overlap RS > 1.5 baseline separation Resolution for separated peaks R_S=(2(t_(R,2)-t_(R,1)))/((w_(b,1)+w_(b,2))) t = retention times of 2 peaks w = widths of 2 peaks Resolution for overlapped peaks R_S=1.18 ((t_(R,2)-t_(R,1)))/((w_(1,h/2)+w_(2,h/2))) Column volume R_S=√(N_2 )/4×(α-1)/α×k_2/(k_2+1) Column efficiency; selectivity term; retention time; Retention factor k standardized to dead time & independent of column. Compare basing on column dimensions and flow rate. for unretained analyte k=0 for irreversible bound analyte k=∞ Recommended: 0.5 – 10 – above retention is strong, resolution & selectivity decreased. Selectivity α k=t_R-t_0

Selectivity α described separation of peak max of 2 peaks. High α = better relative separation of 2 peak maxima. Recommended α > 1 To improve column selectivity changed should be MP, SP or MP pH. Selectivity α α=k_2/k_1 =(t_R2-t_0)/(t_R1-t_0 ) Number N of theoretical plates – imaginary column dividers – in those, analytes equilibrate between MP & SP. Measure column efficiency, depends on substance and conditions. The longer the N, the narrower the peaks, the better the efficiency. Method development The goal is to achieve the best resolution in shortest time by changing variables. Start with MP composition (k & α), then SP and column conditions. Changing retention factor k between 0.5 – 20 increase resolution & analysis times; Changing selectivity α increase resolution by shifting peak maxima positions. Changing α change k. Factors influencing k & α: MP & SP compositions Temperature (small effect) Solvent strength – increased would cause better separation due to changed chemical nature – retention time changed. Too much cause too short retention time so also overlap. pH for ionizable analytes – change charge state of the peptides, changing the polarity Changing N – column conditions - increasing narrower peaks with less overlap; reducing dispersion Factors influencing N: Flow rate Column length SP particle size Column & packing materials Column conditions improvement: by homogenous & spherical particles, small pores to get high surface area. Wide pores have reduced surface and sterical hindrance but separate macromolecules like proteins.

Totally porous particles TPP Superficially porous particles SPP Perfusion particles Most common & universal. Give broad peaks due to different diffusion speeds through the particle. Solid core with thin porous shell; give shorter diffusion paths, cause sharp peaks for higher sensitivity, limit sample loads. Small diffusion peak with large pores. No diffusion, flow by flow rate.

Packaging based on silica materials SiO2: High mechanical strength – sustainable High column efficiency due to tailored TPP with different pore sizes Chemical surface able to be modified with functional groups Compatible with aqueous & organic solvents No swelling Stable in pH 3 – 9, degraded by hydrolysis Polar, but chlorosilanes make unpolar for reverse phase. Endcapping of free silanol groups with short alkyl residues to reduce surface silanol population (blocking silanol group – analyte interactions) Hybrid silica material: methyl group replace surface silanol (give stability), bond susceptible to pH hydrolysis, endcapped silanol group. Stable in high pH Lower activity with basic analytes; less tailing Improved peak shape Limited surface area due to limited porosity Packaging based on polymer-based materials: Stable in pH 1 – 13 Have silanol groups, acidic; that make basic analytes make peaks with tailing. LC-MS required water wettable phases to ionise the analytes and make them less hydrophobic. If it is more than 95% water, the phase collapse. Are small, organic acid molecules with hydrophobic & basic properties. Control spacing between bonded ligands – wide them Add selectivity of low energy silanol groups Vicinal silanol groups are hydrated Ligands are used for hydrophobic & hydrophilic interactions with analyte, prevent phase collapse. Have mixed mode of retention and are compatible with LC-MS. Polar embedded phases prevent collapse of C18 ligands, provide selectivity (due to hydrophilic interactions), can be used in 100% aqueous MP. Ionic analytes in rp-HPLC – ipc Henderson-Hasselbalch equation pH=〖pK〗a+log ([conjugate base])/([acid]) A way to handle strongly ionic compounds in RP-HPLC. Retention times of basic & acidic analytes in RP-HPLC depend on the pH value of MP; but the pKa of the analyte is also important. If pH is equal to pKa, 50% of the molecules will be charged and 50% uncharged = lead to broad peak shapes due to equilibrium & make difficult reproducibility; Low pH = most retention; High pH = least retention; 50:50 occurs in unbuffered aqueous MP – require buffers with pKa equal to highest capacity pH. Method development: 1. pH adjusted so the analyte is uncharged; (Acidic require acidic pH; pH < analyte pKa; Basic require alkaline pH; pH > analyte pKa). 2. Difference between pH of MP & pKa of analyte is at least one unit; making 90% uncharged analyte 3. Choose suitable buffer for pH: max buffer capacity if pH = buffer pKa Higher buffer capacity required total buffer concentration between 10-100mM. Ion pair chromatography IPC use RP columns to separate charged analytes dissolved in MP. Allows to simultaneously separate ionised & non-ionised analytes. Ion pair reagents have hydrophobic & hydrophilic parts (amphiphilic structure)– are surfactants. Ion pair use opposite charge to that of ionic analyte to produce uncharged ion pair at the end. Partition model: analyte molecule interacts first with ion pair reagent in the MP. Adsorption model: ion pair reagent react with surface of SP first. First the pH is adjusted so the analyte is fully ionised: acidic analytes require pH > pKa, basic analytes require pH < pKa. Retention time increase with increased concentration of ion pair reagent & with increased alkyl chain length of ion pair reagent. Problem: peak tailing due to uncontrolled ionic interactions with ionised silanol groups at pH > 6; between + charged analytes & - charged analytes. Reduced with Endcapping, pH change (remove ionisation), increased buffer concentration (mask silanol interactions), modify MP with TEA (interact with ionised silanol instead of analyte). Tailing factor T_f=W(0.05)/2f

Reverse phase chromatography Non-polar SP, polar MP Separation principle is the retention based on hydrophobicity, used for bioorganic molecules (peptides, proteins). Eluotropic series have the solvent strength adjusted with water & co-solvents (methanol, ACN) to change retention & selectivity. Used to optimise NPC. Lower viscosity = higher separation, lower pressure. UV absorption < 190 nm Toxic THF as co-solvent Minor polarity, miscible with water Slow column regeneration Reactive with oxygen Critical resolution on chromatogram describes resolution between two worst resolved peaks. Typical MP is water + methanol / CAN Selectivity for non-ionic samples depends on: Mobile phase composition (best way) Stationary phase is unpolar bonded phases on silica support or polymer based.

Stationery phase composition – column type Temperature – no large effect on selectivity, but 1 ºC decreases k by 1-2% Gradient hplc Mobile phase is modified during the run by increasing solvent strength over time (reducing water content). Allow for shorter analysis times while retaining baseline separations. Used for samples with wide k-value ranges & for protein samples (improved reproducibility for proteins); at higher % proteins are denaturated = improved reproducibility for proteins. Softening the gradient = higher resolution + high retention time.

Method development: start with fast-linear gradient with max. 6 column types. Best combination determined by most peaks & their shape. Then modifications of organic solvent in MP as a gradient; lastly eluents combination, pH, column types, temperature, gradient steepness, temperature, column length. Normal phase chromatography npc Opposite to RP-HPLC: works with unpolar MP (hexane, isooctane) and polar SP (silica beads, polar bonded phases on silica support). Produce opposite to RP-HPLC elution orders due to opposite polarity. Used for bioanalytical application – separation of fat-soluble vitamins. Sample should be insoluble in aqueous systems, unretained / too strongly retained by RP-HPLC, do unsatisfactory band spacing with RP-HPLC Optimisation with columns of smaller particle diameters (for higher retention factors), solvent strength based on eluotropic series for NPC: (tert. butanol > toluene > isooctane > n-hexane); solvent strength cannot be too high (polar solvents interact strongly with surface of SP) Band broadening Sample plug starts with rectangular shape and broadener & dilute during separation. When getting broader theoretical plate increase. Van- Deemter equation H=A+B/u+C×u H = height, measure band broadening, equal to N u = linear flow rate [cm/s] L = column length N = theoretical plates H=L/N Contributing factors: A-term: flow rate independent, describes Multipath effect (Eddy diffusion). Describes how molecules of the same kind have different pathways through the column due to particle size & shape; giving different retention times. Use homogenous column beds with smaller & spherical beads to counteract. B-term: pronounced at lower flow rates, described longitudinal diffusion. Plug diffuses parallel to the column axis becoming broaden. Counteracted by higher flow rate & solvent viscosity. C-term: pronounced at higher flow rates, described mass transfer between MP & SP. Strong at higher flow rates as the analytes have no time to interact with SP particles = less time to average the differences. Counteracted by small TPP or core-shell particles for shorter diffusion times; higher temperature to give low viscosity and increase mass transfer; lowering flow rate.